Research on the Effects of NO 3 -N, CO 2, O 2 x Swimming Speed, and Strain x Photoperiod on Salmonid Performance, Health, and Welfare

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1 Good C, Davidson J, Waldrop T, Snekvik K, Kenney PB, Mazik P, McCormick S, Shepherd B, Wolters W, Baeverfjord G, Takle H, Terjesen B, & Summerfelt S Research on the Effects of NO 3 -N, CO 2, O 2 x Swimming Speed, and Strain x Photoperiod on Salmonid Performance, Health, and Welfare

2 Research at The Freshwater Institute

3 Closed Containment Facilities with Water Recirculation

4 Recent Research at The Freshwater Institute Atlantic salmon Salmo salar growout in freshwater closed-containment systems: (1) effects of strain and photoperiod manipulation (2) effects of high and low dissolved carbon dioxide (3) effects of swimming speed and dissolved oxygen

5 Current Research at The Freshwater Institute Rainbow trout Oncorhynchus mykiss in freshwater closed-containment systems: Effects of high vs. low NO 3 -N

6 Current Research at The Freshwater Institute STUDY 1 Effects of strain and photoperiod manipulation on Atlantic salmon

7 Objectives Determine how Atlantic salmon strain and photoperiod manipulation (to produce smoltification) influence growth, processing attributes, and sexual maturity to 26 months post-hatch in freshwater RAS.

8 Materials and Methods 2X2 Factorial study Two Atlantic salmon strains: 1. St. John s 2. Gaspe Two early rearing light regimes: 1. Continuous light 2. Continuous light with S 0 winter 12h light, 12h dark for six weeks, then return to constant light

9 Seawater Challenge Post-Photoperiod Manipulation

10 Three 10 m 3 culture tanks in one freshwater reuse system Both strains and photoperiod treatment fish co-mingled within each tank for n=3 replication

11 Results Density range = 50 kg/m kg/m 3 Good overall feed conversion throughout study 1.05 with 40:30 diet Overall survival >95% No vaccinations or treatments necessary, aside from occasional salt to control fungus Gaspe strain outperformed St. John s at 26 months post-hatch

12 Mean Fish Weight to 26 months Gaspe (Continuous Light + S0 Winter) Gaspe (Continous Light) St. John's (Continuous Light + S0 Winter) St. John's (Continuous Light) 3782 ± 92 g 3527 ± 116 g 4262 ± 103 g 3879 ± 149 g Weight (g) Days Post-hatch

13 Gaspe St. John s Variable S0 Winter Constant S0 Winter Constant Weight (g) *** 4262 ± ± ± ± 116 Dress Yield (%) 89.8 ± ± ± ± 0.5 Fillet Moisture (%) 62.7 ± ± ± ± 0.7 Fillet Fat (%)*** 17.2 ± ± ± ± 0.8 Fillet Protein (%) *** 19.7 ± ± ± ± 0.2 Fillet Ash (%) 1.38 ± ± ± ± 0.05 Viscera Index (%) 8.72 ± ± ± ± 0.5 Significantly (p<0.05) different between strains *** Significantly (p<0.05) different between both strain and photoperiod treatment

14 Current Research at The Freshwater Institute STUDY 2 Long-term effects of high (20 mg/l) vs. low (10 mg/l) CO 2 exposure on Atlantic salmon

15 Background: Dissolved CO 2 Inverse relationship of CO 2 with ph Long-term exposure to elevated CO 2 Decreased hemoglobin oxygen binding capacity (Bohr effect) Increased ventilation, elevated blood pressure Reduced growth rate Higher FCR Nephrocalcinosis Also, increased solubility of toxic metals at lower water ph

16 Background: Dissolved CO 2 Growth of salmonids significantly reduced at 30 mg CO 2 / L Maximum limit < 20 mg CO 2 / L Recent review suggests 10 mg CO 2 / L max for Atlantic salmon

17 Background: Dissolved CO 2 Aside from fish health implications. Economic Considerations Decreasing tank CO 2 concentrations requires pumping more water flow & installing a larger stripping unit (cascade column or aerated basin) increases fixed costs increases variable costs to pump water

18 Background: Dissolved CO Rainbow trout Similar study completed with rainbow trout (Good et al Aquacultural Engineering) Weight (grams) mg/l CO2 10 mg/l CO Days Post-hatch

19 Atlantic salmon growout in freshwater: - Six replicated RAS - 3 with 20 mg/l CO 2-3 with 10 mg/l CO 2 - Alkalinity >200 mg/l - 100% DO saturation

20 Performance - Growth Final weight 807 days post-hatch High CO 2 : 2879 ± 35 Low CO 2 : 2896 ± 12 Equal salmon growth at 10 and 20 mg/l of CO 2

21 Performance - TGC

22 Survival Mean survival (%) High CO 2 : 99.2 ± 0.3 Low CO 2 : 99.1 ± 0.3 Culls due to fungus (%) High CO 2 : 3.75 ± 1.05 Low CO 2 : 3.41 ± 1.27 * approx lbs of salt added to each system during study to control fungus

23 Feed Conversion

24 Parameter CO Mean ± SE p-value Sodium (mmol/l) 2 High 148 ± Low 152 ± 1.59 Chloride (mmol/l) High 127 ± Low 132 ± 0.89 Calcium (mmol/l) High 1.52 ± Low 1.57 ± 0.01 Glucose (mg/dl) High 66.0 ± Low 71.6 ± 1.99 Hematocrit (%PCV) High 33.7 ± Low 39.7 ± 2.17 Hemoglobin (g/dl) High 11.5 ± Low 13.5 ± 0.74 ph High 7.11 ± Low 7.06 ± 0.01 pco 2 (mmhg) High 61.8 ± Low 48.7 ± 1.17 Bicarbonate (mmol/l) High 19.6 ± 0.26 <0.001 Low 13.7 ± 0.26 Total CO 2 (mmol/l) High 21.4 ± 0.27 <0.001 Low 15.2 ± 0.34

25 ANOVA p-values 20 mg/l CO 2 1-wk 20 mg/l CO 2 3-wks 10 mg/l CO 2 1-wk 20 mg/l CO 2 3-wks mg/l CO 2 1-wk mg/l CO 2 3-wks

26 Current Research at The Freshwater Institute STUDY 3 Effects of swimming speed (2 BL/s vs. 0.5 BL/s) and dissolved oxygen (100% vs. 70% saturation) on Atlantic salmon

27 Published research on salmonids suggests prolonged exercise leads to: Increased growth Better feed conversion Less size variation at harvest Improved disease resistance Swimming Speed Reduced aggressive behavior Improved flesh texture Increased oxygen consumption

28 Dissolved Oxygen Can become limiting as production levels, feeding, and biomass increases Low dissolved oxygen can lead to: Slower growth Poor feed conversion Increased disease Decreased swimming fitness

29 Swimming Speed X Dissolved Oxygen Recommended for salmonid aquaculture: Body-lengths/s swimming speed 100% oxygen saturation Difficult to achieve in raceways; relatively straightforward in circular tanks Plenty of research on exercise and DO, but little work in assessing these parameters in combination

30 2X2 Factorial Study Design Two swimming speeds: <0.5 (BL/s) (BL/s) Two dissolved oxygen levels: High: 100% saturation Low: 70% saturation

31 Materials and Methods Identical Flow-Through Circular Tanks 12 tanks total: 3 replicates of each Swimming speed /DO combination (random)

32 BL/s 70 % DO 0.5 BL/s 100% DO Performance Weight (g) BL/s 70% DO 2 BL/s 100% DO Days Post-Hatch

33 DO 100% saturation DO 70% saturation 2 BL/s 0.5 BL/s 2 BL/s 0.5 BL/s Weight (g) ± ± ± ± 6.9 Treatment df F p-value Swimming speed Dissolved oxygen < Swimming speed X dissolved oxygen

34 Precocious Males: 2 BL/sec : 6.4% < 0.5 BL/sec: 11.5% Logistic regression model reporting odds ratios for the probability of precocious males within each treatment group: Treatment Odds ratio (95% conf. int.) p-value 0.5 BL/s (1.121, 3.208) % DO (0.546, 1.636) 0.839

35 Additional assessments: Cardiosomatic index higher in high DO group (p=0.059) No difference in visceral index

36 Assessing the effects of elevated nitrate nitrogen on the performance, health, and welfare of juvenile rainbow trout in recirculating aquaculture systems John Davidson, Christopher Good, Kevin Snekvik Carla Welsh, and Steven Summerfelt

37 Introduction Low exchange recirculating aquaculture systems (RAS) are being utilized more often due to: Limited clean water resources Need for a controlled culture environment Biosecurity Improved treatment of smaller concentrated waste flows Very little known regarding effects of accumulating water quality constituents on fish when RAS are operated at low water exchange rates.

38 Preliminary Research Two separate studies were conducted in 6 replicated RAS 1) Low exchange vs. high exchange 2) Very low exchange vs. near-zero exchange Study 1 Feed Loading Rate (kg feed/m 3 makeup water/ day) Low Water Exchange High Water Exchange Hydraulic Retention Time (days) Study 2 Feed Loading Rate (kg feed/m 3 makeup water/ day) Near-Zero Water Exchange Very Low Water Exchange > 71 < 44 Hydraulic Retention Time (days) > 103 < 36

39 Recirculating System (9.5 m 3 ) 5.3 m 3 Dual-drain tank Radial flow settler Drum filter (60 µm screens) Pump sump 1-HP centrifugal pump Heat Exchanger Fluidized Sand Biofilter Low Head Oxygenator (LHO) CO 2 Stripping Column Ozone Generators (3 RAS)

40 Swimming Speed Study 1

41 Swimming Behavior Study 1 Signficantly increased side swimming behavior in low exchange RAS Low Exchange High Exchange

42 Side Swimming Study 1

43 Deformities and Survival Study 2 Survival Very Low Exchange (HRT < 36 days) ± 0.4% Near-Zero Exchange (HRT > 103 days) ± 1.9% Increased spinal deformities observed in near-zero exchange RAS ASSESSING THE EFFECTS OF ELEVATED NITRATE NITROGEN ON THE PERFORMANCE, HEALTH, AND WELFARE OF JUVENILE RAINBOW TROUT Oncorhynchus mykiss IN WATER RECIRCULATION AQUACULTURE SYSTEMS

44 Water Quality Results Many water quality parameters were assessed Only a few existed at potentially harmful concentrations that correlated with fish health and welfare problems Nitrate nitrogen Dissolved potassium Dissolved copper Davidson et al., Abnormal swimming behavior and increased deformities in rainbow trout cultured in low exchange water recirculating aquaculture systems. Aquacultural Engineering 45,

45 Nitrate Study Methods Rainbow trout stocked equally amongst 6 RAS Mean weight = grams Six RAS were operated at equal flushing rates Enough flushing to limit accumulation of other constituents Hydraulic Retention Time = 1.3 days Feed Loading Rate = 0.72 kg feed/m 3 makeup water/day Sodium nitrate continuously dosed to 3 RAS using peristaltic pumps to create high nitrate nitrogen Target NO 3 -N mg/l Sodium Sulfate continuously dosed to other 3 RAS to maintain similar conductivity and Na + concentrations in all RAS Target NO 3 -N < 30 mg/l

46 Water Quality - Nitrogen Parameter (mg/l) High NO 3 -N Low NO 3 -N Nitrate Nitrogen 29 ± 0 89 ± 0 Total Ammonia Nitrogen 0.35 ± ± 0.02 Unionized Ammonia ± ± Nitrite Nitrogen 0.02 ± ± 0.00

47 Water Quality Parameter (mg/l) High NO 3 -N Low NO 3 -N Conductivity 1176 ± ± 7 Alkalinity 195 ± ± 0 Hardness (as CaCO 3 ) 306 ± ± 2 Carbon Dioxide 13 ± 0 13 ± 0

48 Water Quality - Metals Parameter (mg/l) High NO 3 -N Low NO 3 -N Calcium 114 ± ± 0 Potassium 16.0 ± ± 0.1 Sodium 107 ± ± 5 Sulfur 12 ± 0 88 ± 2

49 Growth Performance

50 Feed Conversion

51 Survival

52 Side Swimming

53 Other Results Swimming speed was generally greater for the high nitrate treatment, but not always significantly There were very few deformities in general and no significant difference in number of spinal deformities between treatments Tissue and blood samples were sent out for histopathology analysis and results are pending

54 Conclusions Nitrate nitrogen from mg/l appears to cause chronic effects to juvenile rainbow trout Slightly decreased growth and FCR Unusual behavior side swimming Decreased survival Low exchange RAS can be operated/ designed with: Limits for NO 3 -N so that water exchange/ feed loading maintain NO 3 -N within safe limits With denitrification unit processes that remove NO 3 -N Be aware that nitrate nitrogen could cause chronic effects to cultured species at much lower concentrations than reported by some literature sources

55 Acknowledgements All research supported by the Agriculture Research Service of the United States Department of Agriculture, under Agreement No Opinions, conclusions, and recommendations are of the authors and do not necessarily reflect the view of the USDA. All experimental protocols were in compliance with Animal Welfare Act (9CFR) and have been approved by the Freshwater Institute Animal Care and Use Committee. Special thanks to Karen Schroyer, Christine Marshall, Susan Glenn, Susan Clements for water quality analysis and technical assistance.